Background and purpose
Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immunomediated condition affecting the peripheral nervous system where probably macrophages are the ...primary effector cells for demyelination. Reactive oxygen species (ROS), catalyzed by the NOX family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, can induce peroxidation and are potentially injurious to myelin. Our aim was to assess the activity of NOX2, an isoform of NOX, in a series of CIDP patients and to analyze the effect of intravenous immunoglobulin (IVIg) on NOX2.
Methods
Thirty CIDP patients treated with IVIg and 30 control subjects were enrolled. To evaluate NOX2 activity, neutrophil and monocyte oxidative burst was measured directly in fresh whole blood using the Phagoburst™ assay, a fluorescence‐activated cell sorting method. The mean fluorescence intensity, emitted in response to different stimuli, leads to the production of ROS and corresponds to the percentage of oxidizing cells and their enzymatic activity.
Results
Mean fluorescence intensity values for granulocyte and monocyte burst in patients (mean 633.3, SD 191; mean 111.8, SD 28.5) were different from those measured in healthy controls (granulocytes, mean 436.6, SD 137.0, P = 0.0003; monocytes, mean 78.2, SD 17.3, P = 0.000001). Moreover, IVIg administration increased both granulocyte (P = 0.005) and monocyte (P = 0.0009) burst.
Conclusion
Our findings demonstrate that oxidative burst is significantly increased in CIDP patients and that treatment with IVIg enhances oxidative values, thus representing a possible IVIg therapeutic effect linked to a regulatory effect of ROS. Based on this, the development of treatments targeting the specific activation of NOX may be beneficial in autoimmune disorders.
SARS-CoV-2 has resulted in high levels of morbidity and mortality world-wide, and severe complications can occur in older populations. Humoral immunity induced by authorized vaccines wanes within 6 ...months, and frequent boosts may only offer transient protection. GRT-R910 is an investigational self-amplifying mRNA (samRNA)-based SARS-CoV-2 vaccine delivering full-length Spike and selected conserved non-Spike T cell epitopes. This study reports interim analyses for a phase I open-label dose-escalation trial evaluating GRT-R910 in previously vaccinated healthy older adults (NCT05148962). Primary endpoints of safety and tolerability were assessed. Most solicited local and systemic adverse events (AEs) following GRT-R910 dosing were mild to moderate and transient, and no treatment-related serious AEs were observed. The secondary endpoint of immunogenicity was assessed via IgG binding assays, neutralization assays, interferon-gamma ELISpot, and intracellular cytokine staining. Neutralizing antibody titers against ancestral Spike and variants of concern were boosted or induced by GRT-R910 and, contrasting to authorized vaccines, persisted through at least 6 months after the booster dose. GRT-R910 increased and/or broadened functional Spike-specific T cell responses and primed functional T cell responses to conserved non-Spike epitopes. This study is limited due to small sample size, and additional data from ongoing studies will be required to corroborate these interim findings.
IntroductionAmyotrophic lateral sclerosis (ALS) is a fatal progressive neurological disorder characterised by a selective degeneration of motor neurons (MNs). Stem cell transplantation is considered ...as a promising strategy in neurological disorders therapy and the possibility of inducing bone marrow cells (BMCs) to circulate in the peripheral blood is suggested to investigate stem cells migration in degenerated ALS nerve tissues where potentially repair MN damage. Granulocyte-colony stimulating factor (G-CSF) is a growth factor which stimulates haematopoietic progenitor cells, mobilises BMCs into injured brain and it is itself a neurotrophic factor for MN. G-CSF safety in humans has been demonstrated and many observations suggest that it may affect neural cells. Therefore, we decided to use G-CSF to mobilise BMCs into the peripheral circulation in patients with ALS, planning a clinical trial to evaluate the effect of G-CSF administration in ALS patients compared with placebo.Methods and analysisSTEMALS-II is a phase II multicentre, randomised double-blind, placebo-controlled, parallel group clinical trial on G-CSF (filgrastim) and mannitol in ALS patients. Specifically, we investigate safety, tolerability and efficacy of four repeated courses of intravenous G-CSF and mannitol administered in 76 ALS patients in comparison with placebo (indistinguishable glucose solution 5%). We determine increase of G-CSF levels in serum and cerebrospinal fluid as CD34+ cells and leucocyte count after treatment; reduction in ALS Functional Rating Scale-Revised Score, forced vital capacity, Scale for Testing Muscle Strength Score and quality of life; the adverse events/reactions during the treatment; changes in neuroinflammation biomarkers before and after treatment.Ethics and disseminationThe study protocol was approved by the Ethics Committee of Azienda Ospedaliera Universitaria ‘Città della Salute e della Scienza’, Torino, Italy. Results will be presented during scientific symposia or published in scientific journals.Trial registration numberEudract 2014-002228-28.
Abstract
Mutations in the KRAS gene are among the most common driver mutations in cancer. Targeting KRAS G12C mutations has shown some promise in clinical studies, though acquired resistance ...mutations pose a challenge for small molecule-based monotherapies. Vaccines targeting KRAS mutations remain highly desirable for durable tumor control and clinical benefit to patients with solid tumors. While cytotoxic CD8 T cells are critical to tumor control and clearance, CD4 T cell activation is a key component of a durable anti-tumor response. Cancer neoepitopes with homology to infectious diseases antigens (mimotopes) were associated with long-term clinical benefit following CTLA-4 blockade. In this study, we screened healthy donor PBMC samples via ex vivo interferon-gamma (IFNγ) ELISpot and identified KRAS G12C-specific T cell responses associated with cross-reactive TCRs recognizing a bacterial lipoprotein peptide sequence with homology to KRAS G12C. Healthy donors with T cell responses to both KRAS G12C and mimotope peptides shared common HLA-DR alleles, and KRAS G12C-specific T cell responses were CD4 dependent via depletion assays. KRAS G12C-reactive CD4 healthy donor T cells were polyfunctional as assessed by intracellular cytokine staining, and killing of class II target cells presenting G12C peptides assessed by Incucyte assay was CD4-dependent. We further identified vaccine-induced KRAS G12C specific CD4 T cell responses in a patient with KRAS G12C-positive non-small cell lung cancer (NSCLC) receiving a shared neoantigen-targeting cancer vaccine consisting of Chimpanzee adenovirus prime and self-amplifying mRNA (samRNA) boost vaccinations in combination with nivolumab 480 mg IV (NCT03953235). This patient had previously progressed on prior checkpoint inhibitor therapy and showed signs of clinical benefit with molecular response (e.g., ctDNA reduction) and tumor shrinkage following vaccination. Patient PBMCs were analyzed by ex vivo IFNγ ELISpot, and G12C-specific T cell responses were detectable at baseline and increased following vaccination. T cell receptor (TCR) and transcriptome single cell sequencing analyses showed a large clonal population of CD4 effector memory cells in the post-vaccination samples. Functional analyses of patient-derived TCR clonotypes from post-vaccination samples via recombinant TCR screening assays identified two functional clonotypes recognizing KRAS G12C epitopes in the context of HLA-DR on target cells. Studies to further elucidate KRAS G12C vaccine-induced TCR functionality in primary cells and transgenic mouse models are ongoing. This study provides important insight into both naturally occurring bacterial mimotope-based and vaccine-induced T cell responses against KRAS G12C neoepitopes that informs future therapeutic approaches targeting KRAS G12C tumors.
Citation Format: Christine D. Palmer, Meghan G. Hart, Sonia Kounlavouth, Harshni Venkatraman, Lauren D. Kraemer, Martina Marrali, Calixto Dominguez, Fatema Z. Chowdhury, Jason R. Jaroslavsky, Lindsey Arcebuche, Lorenzo Hernandez, Bukola Adeoye, Severino Cuison, Amy R. Rappaport, Greg Boucher, Monica Lane, Melissa Rotunno, Kenneth Avocetien, Leiliane Sousa, Chris Puccia, Molly Likes, Rahul Vegesna, Rita Zhou, Alexis Mantilla, Matthew J. Davis, Ankur Dhanik, Melissa Johnson, Andrew R. Ferguson, Karin Jooss. HLA-DR-restricted CD4 T cell responses to KRAS G12C in healthy donors linked to bacterial mimotope: lessons for KRAS neoantigen vaccines in cancer patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2267.
Abstract
The immunogenicity and efficacy of RNA-based vaccine platforms has been abundantly shown through their application in prophylactic SARS-CoV2 vaccines. Contrasting to mRNA based vectors, self ...amplifying mRNA platforms may offer dose-sparing and superior induction of T cell responses, and may also trigger distinct innate immune pathways, which may exert adjuvanting or inhibiting effects on vaccine-induced immunity. Optimal dosing for a novel self-amplifying mRNA (SAM) in a heterologous prime-boost vaccination approach consisting of Chimpanzee Adenovirus (ChAd) prime and SAM boosts was evaluated in two first-in-human phase 1/2 clinical trials assessing personalized neoantigen vaccines in patients with metastatic cancer (NCT03639714, NCT03953235). SAM vaccine dose escalation was performed to assess safety, tolerability, and immunogenicity, including administration of up to 8 SAM doses at 30, 100, or 300µg following a fixed dose of ChAd (1012 vp) over the course of a year. SAM was safe and well tolerated at all 3 dose levels, with no evidence of increasing reactogenicity with sequential doses. However, while immune monitoring via IFNγ ELISpot revealed that the 30µg SAM dose boosted T cell responses induced by the ChAd prime, the 100µg and 300µg SAM doses resulted in maintenance of T cell levels, without a clear T cell boost, suggesting a non-linear and likely bell-shaped dose-response curve to SAM in humans. Follow-up studies in non-human primates (NHPs) using a model antigen revealed dose-dependent increases in serum IFNa levels following administration of increasing SAM doses. Similarly, while multiple inflammatory cytokines were transiently increased following both ChAd and SAM administration in patients, serum IFNa levels were only increased 24h post SAM administration and correlated positively with SAM dose. Increased IFNa levels post SAM dosing suggested activation of mRNA-sensing innate immune pathways that may reduce the amplification of, and/or antigen expression by, the SAM vector and thus blunt T cell boosting at higher SAM doses. In addition, analysis of T cell responses in patients and NHPs showed increased boosting of T cell responses with longer intervals. These data lead to a reduction of the SAM dose to 30µg and adjusting SAM dosing intervals to 8 weeks in the Phase 2 portion of these clinical studies. Multiple patients have been dosed with the adjusted vaccine regimen, and preliminary data suggest robust boosting of ChAd-induced neoantigen-specific T cell responses with the selected SAM dosing regimen and the 30µg dose. We anticipate that this translational approach of adjusting clinical vaccine regimens based on strong translational immune data will increase the potency of our heterologous neoantigen vaccine, and subsequently provide more durable clinical benefit to patients with cancer.
Citation Format: Christine D. Palmer, Amy R. Rappaport, Meghan G. Hart, Lauren D. Kraemer, Sonia Kounlavouth, Martina Marrali, Jason R. Jaroslavsky, Charmaine N. Nganje, Annie Shen, Gregory R. Boucher, Melissa A. Kachura, Ciaran D. Scallan, Sue-Jean Hong, Leonid Gitlin, Alexander I. Spira, Chrisann Kyi, Daniel V. Catenacci, Raphael Rousseau, Andrew Ferguson, Karin Jooss. Lower doses of self-amplifying mRNA drive superior neoantigen-specific CD8 T cell responses in cancer patients versus high doses abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4159.
Abstract
Vaccines targeting neoantigens identified from common tumor driver mutations are of increasing interest as evidence of clinical benefit builds, and opportunities to combine such vaccines ...with immune modulators are growing. Our individualized neoantigen vaccine (GRANITE) has shown clinical benefit and strong, consistent CD8 T cell induction in patients. We have developed an analogous off-the-shelf product (SLATE) targeting shared neoantigens that offers manufacturing simplicity and faster administration to patients with shared driver mutations. A Phase 1/2 trial of a heterologous prime/boost vaccine regimen using a chimpanzee adenovirus (ChAd) prime and self amplifying mRNA (SAM) boosts (SLATE, NCT03953235) was initiated to assess safety, tolerability, and immunogenicity in patients with advanced cancers. SLATE version 1 encodes 20 unique neoantigens to various shared driver mutations (KRAS, TP53, etc.). Patients were selected if their tumors harbored one of the 20 neoantigens encoded by the vaccine cassette and an HLA Class I allele that presents that neoantigen. Administration of ChAd prime and repeated administration of 30, 100, or 300µg SAM doses were safe and well tolerated in all subjects dosed (n=26), with no evidence of increasing reactogenicity with sequential dosing. Early efficacy signals (molecular responses; one unconfirmed RECIST response) were observed in NSCLC subjects all treated with and progressed on prior anti-PD(L)1. Analysis of T cell responses pre and post immunizations by ex vivo IFNγ ELISpot did not show robust responses to KRAS neoantigens across all patients. However, objective CD8 T cell responses to KRAS antigens post vaccination were detectable after in vitro stimulation, suggesting the induction of low-level KRAS specific T cell responses in vivo. In contrast, HLA-matched responses to TP53 neoantigens encoded by the vaccine were consistently detected via ex vivo ELISpot in these same patients. These data suggests that an immunodominant T cell response to the TP53 mutations may have outcompeted the response to the less immunogenic KRAS mutations restricted and presented by the same HLA in vivo. Differential surface peptide-HLA (pHLA) density may explain these discordant findings, and subsequent targeted mass spectrometry analyses revealed detection of TP53 pHLA complexes at a higher frequency compared to KRAS mutations in single HLA-allele cell lines. Redesigned vaccine cassettes excluding the TP53 epitopes and repeating KRAS epitopes demonstrated increased immune responses (ex vivo IFNγ ELISpot) compared to cassette version 1 in HLA transgenic mice, further supporting the tumor neoantigen immunodominance hierarchy observed in humans dosed with SLATE version 1. A re-designed product (SLATE v2) focusing exclusively on KRAS mutations (G12C, G12D, G12V and Q61H) is currently being assessed in phase 2 in patients with advanced KRAS-driven tumors.
Citation Format: Amy R. Rappaport, Christine D. Palmer, Annie Shen, Claudia X. Dominguez, Meghan G. Hart, Lauren D. Kraemer, Sonia Koulavouth, Martina Marrali, Jason R. Jaroslavsky, Charmaine N. Nganje, Ciaran D. Scallan, Sue-Jean Hong, Leonid Gitlin, Monica Lane, Daniel V. Catenacci, Chrisann Kyi, David P. Carbone, Hossein Borghaei, Raphael Rousseau, Andrew Ferguson, Karin Jooss. Optimization of shared neoantigen vaccine design to increase vaccine potency: From bench to bedside and back abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3578.
The paper reports two cases of transitory hyperphosphatasemia in two siblings of 13 and 29 months without modifications of the liver, kidney or bones. Laboratory data are given for hepatic and renal ...function and for the calcium/phosphorus metabolism. The paper underlines the observation of rotavirus and the consequent clinical symptoms in the two subjects. The hypothesised etiopathogenesis of transitory idiopathic hyperphosphatasemia is referred to a viral mechanism, and to rotavirus in particular.
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